Isolation of an organic sulfur compound from asparagus



Patented July 10, 1951 ISOLATION OF AN ORGANIC SULFUR COMPOUND FROMASEARAGUS Eugene F. J ansen, Berkeley, (lalif assignor to the UnitedStates of :America as represented by the Secretary of Agriculture NoDrawing. Original application April 15, 1949,

Serial No. 87,823. Divided and. this'application April14, 1950,. SerialNo. 156,034

(Granted under the act of March 3, 1883,. as amended April 30, 1928; 3700. G. 757) 6 Claims.

This application is made under the act of March 3,1883, as amended bythe act of April 30, 19 28, and the invention herein described, if,patented in any country, may be manufactured and used by or for theGovernment of the United Statesof America for governmental purposesthroughout the world without the payment to me of any royalty thereon.

This application is a division of my co-pending patent applicationSerial No. 87,823, filed April 15, 1949, issued January 30, 1951, asPatent No. 2,539,428.

invention relates to organic sulphur compounds and has among its objectsthe provision of processes for isolating an organic disulphide compoundcontained in asparagus. Other objects will be apparent from thedescription of the invention.

One phase of this invention involves the provision of a process forisolating an organic disulphide compound from asparagus.- This comound,the existence of which has not been. heretofore known, is useful as anintermediate for chemical sytheses and for other purposes as will appearhereinafter. The process by which this all impurities.

disulphide compound is isolated involves extractin'g asparagus juicewith an organic polar solvent whereby a polar solvent extract isobtained which "contains the disulphide compound together with someimpurities. This extract is then further treated to separate the desiredcompound from the impurities. Ithas been found that the disulphidecompound has a carboxylic group and in its acid form is preferentiallysoluble in organic solvents, whereas in its salified (neutrali'zed) formit is preferentially soluble in water. Therefore, further purificationis 'efiected by carrying out one or more transfers from solution inorganic solvent to solution in water and vice versa. In transferringfrom organic solvent to water, basic conditions are employed, whereasfor the reverse transfer, acidic conditions are employed. The number oftransfers to be carried out depends, of course, on the degree of puritydesired. For some purposes, it is sufficient to transfer the compoundfrom organic solvent to water and then back'to organic solvent, and thenevaporate the solvent to obtain the product. In

other cases'where hig hly purified product is desired, the transfers canbe repeated to eliminate For complete and exhaustive purification, thefollowing procedure is used: Asparagus juice is extracted with a polarorganic solvent whereby a polar solvent extract is 011- tained whichcontains the desired organic disulphide compound plus'impurities such.as phe nolic compounds, non acidic compounds, and so forth. This extractis then extracted with an aqueous alkaline solution. By this treatmentthe disulphide compound is transferred to the aqueous phase, but variousimpurities, particularly phenolic compounds, remain in the solventphase. The aqueous extract is then acidifiedand extracted with a polarorganic solvent whereby the disulphide compound is transferred 'to thesolvent phase and a further elimination of lin purities is effected.The. solvent phase is then concentrated, preferably under vacuum, to mamove all the water therefrom. The concentrated solution is then mixedwith an excess of an 'organic fat solvent whereby impurities low insulphur content are precipitated. The disulphide compound is againtransferred to aqueous solu tion by extraction with aqueous alkalinesolution and this aqueous phase, after acidification, is extracted withan organic fat solvent. The resulting extract contains the disulphidecompound in substantially pure form and it can be recovered in solidform by evaporation of the solvent.

In the foregoing isolation technique many alternative materials may beused. For example, butanol (i. e., normal butyl alcohol) is thepreferred polar solvent; however, other polar solvents can be employedwhich are miscible with water to the extent of about 5 percent to about20 percent, as for example, isobutyl alcohol, secondary butyl alcohol,n-amyl alcohol, isoamyl alcohol, and so forth.

"sulphuric, phosphoric, acetic, and so .forth.

As the organic fat solvent, benzene is preferred, but toluene, xylenes,carbon tetrachloride, chloroform, ethylene dichloride, ether, hexane,octane, and mixtures of hydrocarbons such as gasoline, petroleum ether,Stoddard solvent, benzine, naphtha, and other organic fat solvents maybe used.

The disulphide compound isolated from asparagus juice is a yellowamorphous material which containsthe radical; 1

carboxylic radical. The material tends to polymerize on standing eitheras such-or'fin solution, becoming insoluble in organic solvents.

The organic disulphide compound isolated in accordance with thisinvention is useful as an intermediate'for the preparation of diversetypes or derivatives. Thus, for example, it may be used to prepare3,3-dithiolisobutyric acid, a compound useful as an anti-Lewisite agentand as a regulater for enzymic reactions. In preparing 3,3-dithiolisobutyric acid, the organic disulphide compound is subjected toa reduction process. The preferred technique involves dissolving theasparagus disulphide compound in a suitable inei't solvent, as forexample, anhydrous liquid ammonia; and then reducing it with an alkalimetal such' as'so'dium or potassium. The alkali metal derivative of the'3,3'-dithiolisobutyric acid so formed is then converted into the aciditself by addition of a mineral acid such as hydrochloric, sulphuric;and so forth. If it is desired to obtain a purified product, theacidified reaction mass is extracted'withan inert orgam'e'solvent suchas benzene, toluene, xylenes,- petroleum naphtha,

hexane, and s'oforth, and the dithiol acid recovered therefrom byconcentrating and cooling. This procedureis shown in Example II herein,and the reactions involved in this synthesis are illustrated by thefollowing equation NaSCH Asparagus Na acid disulphide .CHC O ONacompound ,liq. NH;

' Hsom CHC O OH HSOz 3,3'-dithiolisobutyric acid can be converted intoits salts by reaction with suitable hydroxides, carbonates, or otherinorganic compounds. For example, the sodium salt of the dithiol acidcan be prepared by reacting it with sodium hydroxide or sodiumcarbonate. In the same manner any desired salt can be prepared by theusual neutralization or metathetic techniques. Salt formationis notlimited to inorganic salts as salts may aIso be made with organic basessuch as amines, quaternary ammonium compounds, ternary sulphoniumcompounds, for example, methyl 'amihe, triethanol amine, glyceryl amine,aniline, morpholinejbrucine, strychnine, pyridine, piperidine,tetraethyl ammonium hydroxide; dodecyl .trimethyl ammonium hydroxide,triethyl sulphonium hydroxide, and so forth.

The synthesis of many other types of organic compounds from theasparagus disulphide compound is disclosed in my prior applicationSerial No. 87,823, filed April 15, 1949, issued as Patent No. 2,539,428.

EXAMPLE I Isolation of the asparagus disulphidccompoand A supply ofasparagus concentrate was procured. This concentrate was prepared byevaporating the juice pressed from asparagus butts until the solidscontent was 62.5 percent. A 9 gallon sample of the concentrate (40kilos) was diluted with an equal weight of water. This solution, whichhad a pH of 4.5, was extracted with '7 gallons of butanol by agitationfor 20 minutes. After standing for 2 hours the liquid was centrifuged,the butanol phase thus being separated from the aqueous phase. Thebutanol phase was then extracted with 3 gallons of dilute aqueous sodiumbicarbonate solution (4 lbs. NaI-ICOa per gallon). The bicarbonateextract was separated and acidified to pH 3 with phosphoric acid, andextracted with 4 gallons of butanol. The butanol extract was evaporatedat 35? C. and undervacuum to a volume of 270 ml. This concentrate wasfiltered and to it was added 1300 ml. of benzene, and the precipitatedmaterial removed by filtration. The butanol-benzene solution was thenextracted with 550 ml. of 5 percent sodium bicarbonate solution; thebicarbonate solution was washed with benzene, and then acidified to pH 2with sulphuric acid. Some sticky black insoluble material was removed byfiltration and centrifugation. The bicarbonate solution was thenextracted with 3 liters of ether and the ether extract dried overanhydrous sodium sulphate and then evaporated to a volume of m1. Thisether solution contained 31.7 grams of the disulphide compound.

EXAMPLE Ii Reduction of the asparagus disulphide compound to3,3'-dithiolisobatyric acid To 175 ml. of liquid ammonia was added 13ml. of the ethereal solution prepared in accordance with Example Iabove, this ether solution containing 4.1 grams of the disulphidecompound.

Metallic sodium was added in small pieces until a relatively permanentblue color was obtained. As the'reduction proceeded the material wentinto solution. The ammonia was allowed to evaporate spontaneously. Theresidue was taken up in 40 ml. of water and quickly acidified withconcentrated hydrochloric acid to pH 2. This aqueous material wasfrozen, then dried under vacuum. The residue was allowed to standovernight with 600 ml. of petroleum ether (B. 1?. 92-100") and thenre-extracted with 200 ml. of petroleum ether. The combinedpetroleum'ether extract was cooled to l8 for several days. Colorlesscrystals of 3,3'-dithiolisobutyric acid separated. Uponrecrystallization from 800 ml. of'petroleum'ether, 1.97 grams-of3,3'-dithiolisobutyric'acid was obtained corresponding to a 48 percentyield on a solid-basis.

The product melted at 6l-62.

Analysis: cinaozsz r Calculated: C, 31.56; H, 5.30; e, 42.12; -mol.-wt.,

Found: C, 32.3; H, 5.30; S, 41.6; neutral equivalent, 152; mol. wt.,155; equivalent weight by -SH titration, 77.

Having thus described the invention, what is claimed is:

l. A process of isolating a disulphide compound contained in asparagusjuice comprising extracting asparagus juice with an alcohol which ismiscible with water to the extent of about 5 percent to about 20percent, extracting the alcohol extract with aqueous alkaline solution,acidifying the resulting aqueous extract and extracting it with anorganic solvent.

2. A process in accordance with claim 1 wherein the alcohol is butanol.

3. A process for isolating an organic disulphide compound contained inasparagus juice which comprises extracting asparagus juice with analcohol which is miscible with water to the extent of about 5 percent toabout 20 percent, extracting the alcohol extract with an aqueousalkaline solution, acidifying the aqueous alkaline extract andextracting it with an alcohol of the aforesaid type, adding an organicfat solvent to the alcohol extract to precipitate impurities, removingsaid impurities, subjecting the fat solvent-alcohol solution to extractwith aqueous alkaline solution, then acidifying the aqueous alkalineextract and extracting it with an organic fat solvent.

4. A process in accordance with claim 3 wherein the alcohol is butanol.

5. A process of isolating a disulphide compound contained in asparagusjuice comprising extracting the asparagus juice with an alcohol which ismiscible with water to the extent of about from 5 percent to 20 percent,thereby obtaining an alcohol extract containing the desired disulphidecompound plus various impurities extracting this alcohol extract with anaque ous alkaline solution thereby transferring the disulphide compoundto the aqueous phase while some of the impurities remain in the alcoholphase, acidifying the aqueous phase and extracting it with an alcoholwhich is miscible with water to the extent of about from 5 percent to 20percent whereby the disulphide compound is transferred to the alcoholphase while other impurities remain in the aqueous phase, concenu poundto an aqueous solution, acidifying this aqueous solution and extractingit with an organic fat solvent thereby transferring the disulphidecompound to the solvent phase, and removing the solvent to recover thedisulphide compound in solid, substantially pure form.

6. A process of isolating a disulphide compound contained in asparagusjuice comprising extracting the asparagus juice with butanol therebyobtaining a butanol extract containing the desired disulphide compoundplus various impurities, extracting this butanol extract with an aqueoussodium bicarbonate solution thereby transferring the disulphide compoundto the aqueous phase while some of the impurities remain in the butanolphase, acidifying the aqueous phase and extracting it with butanolwhereby the disulphide compound is transferred to the butanol phasewhile other impurities remain in the aqueous phase, concentrating thebutanol phase to remove all water therefrom and filtering it, mixing theresulting concentrated filtrate with benzene whereby impurities low insulphur content are precipitated and removed by filtration, extractingthe resulting benzene-butanol filtrate with an aqueous sodiumbicarbonate solution thereby transferring the disulphide compound toaqueous solution, acidifying this aque-- ous solution and extracting itwith ether thereby transferring the disulphide compound to the etherphase, and removing the ether to recover the disulphide compound insolid, substantially pure form.

EUGENE F. JANSEN.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 2,052,219 Dickens Aug. 25, 19362,459,139 Dimick Jan. 18, 1949 2,476,085 Dimick July 12-, 1949 2,481,763Lineweaver Sept. 13, 1949 OTHER REFERENCES 'Schertz: Plant Physiology,3, 211-216 (1928) Methods of Analysis A. O. A. 0., page 794, A. O. A.0., Washington, D. 0., Ed. 6, 1945.

1. A PROCESS OF ISOLATING A DISULPHIDE COMPOUND CONTAINED IN ASPARAGUSJUICE COMPRISING EXTRACTING ASPARAGUS JUICE WITH AN ALCOHOL WHICH ISMISCIBLE WITH WATER TO THE EXTENT OF ABOUT 5 PERCENT TO ABOUT 20PERCENT, EXTRACTING THE ALCOHOL EXTRACT WITH AQUEOUS ALKALINE SOLUTION,ACIDIFYING THE RESULTING AQUEOUS EXTRACT AND EXTRACTING IT WITH ANORGANIC SOLVENT.